Fungal pathogens vary in their pathogenicity, that is, their capacity to cause disease. At one end of the spectrum, highly pathogenic fungi may produce systemic disease when they infect. At the other end of the spectrum, some fungi may establish a more or less permanent residence on, and even in, the superficial body tissue. These fungi are unable to penetrate the body's natural defenses unless these defenses are seriously impaired. Such opportunistic infections have become more common as a byproduct of therapeutic advances and currently pose a significant medical threat to certain classes of individuals or patients. For example, patients receiving antibiotic therapy, undergoing surgical procedures, manipulation of indwelling catheters and those with altered host defense mechanisms due to the use of antibiotics, immunosuppressive drugs or radiation therapy have an increased risk of opportunistic fungal infection. Fungal infections in these already compromised individuals may result in life threatening disease.
Two of the most medically important types of fungi are Candida albicans and Cryptococcus neoformans. Candida albicans is an example of a relatively non-pathogenic yeast (that is, single cell fungus) that is frequently present on normal mucus membranes of the mouth and intestinal tract. Candida albicans generally will not establish an infection in healthy humans but can cause opportunistic infections resulting in chronic local infections in those with compromised host defense mechanisms. Cryptococcus neoformans is especially important because of its predilection for the central nervous system which can cause severe disease in biologically defenseless patients.
The presumptive identification of Candida albicans depends solely upon morphological changes which occur when this fungus is plated and allowed to grow on an appropriate medium. The first morphological change indicative of the presence of Candida albicans is the formation of germ tubes, which appear as tiny appendages extending from the plated unicellular specimens. These germ tubes eventually grow into elongated filaments extending outwardly from a body of the Candida albicans. Formation of germ tubes within 2 to 3 hours after plating of the fungus is presumptive evidence that Candida albicans or Candida stellatoidea are present. In a second stage of growth, generally round bodies appear at the ends of the filaments. These round bodies are known as chlamydospores. Only three species of the Candida genus will form chlamydospores. These are Candida albicans, Candida stellatoidea, and Candida tropicalis. Thus, chlamydospore formation is indicative of the presence of Candida albicans, Candida stellatoidea, or Candida tropicalis.
The differential identification of Candida albicans and Candida stellatoidea is made by a subsequent carbohydrate assimilation test. The basic concept for these tests includes a series of differential media which contain one specific carbohydrate, a nitrogen source and a color dye such as bromcresol purple which is pH sensitive. For yeasts, carbohydrate assimilation is an acidic reaction which can be monitored with pH-indicating dyes, while fermentation involves acid and gas production, requiring a pH-indicating dye and gas trap.
There are two methods presently used for assimilation: (1) a modified Wickerham method and (2) an auxanographic method. In general, the modified Wickerham method uses test tubes of carbohydrate agar media which contain a pH sensitive dye. The tubes are inoculated directly from a yeast subculture plated on a growth medium such as Sabouraud's dextrose agar by transferring a loop of organism to the surface of the carbohydrate agar. Positive assimilation reactions are noticeable in one to seven days. A conventional example of this method includes the inoculation of a test tube of sucrose media containing bromcresol purple with yeast followed by an examination at 24 to 72 hours for a color change of the medium from purple to yellow indicating sucrose assimilation. Candida albicans will give a positive assimilation while Candida stellatoidea is negative. The basic disadvantages are a long time-to-positivity and cumbersome and large storage space requiring specialized racks for storage.
Another conventional plate also utilizes the modified Wickerham method with one basic alteration. The plate is composed of a series of wedges arranged in a circular configuration which contain a carbohydrate media with bromcresol purple, an urea agar and a nitrate media. Instead of a direct inoculum transfer, the yeast is first suspended in sterile water and then transferred by sterile pipet to the various media. The plate is then incubated and examined at 24 hours to 6 days for positive reactions. Disadvantages of this system are long time-to-positivity, relative expense and an increased amount of media used, each well requiring about ten milliliters of media.
The auxanographic method involves a pour plate technique where the yeast is suspended into molten carbohydrate agar medium. The yeast suspension is then poured into a sterile culture (hereinafter sometimes referred to as "petri") plate and allowed to solidify. There are two variations of this method. In one method, the medium does not contain carbohydrate, and after solidification, sterile paper discs impregnated with a specific carbohydrate are placed onto the agar surface of the inoculated pour plate. The carbohydrate then diffuses from the disc into the inoculated medium. Positive assimilation is then measured either by a pH change around the disc in the presence of a pH sensitive dye or by a ring of turbidity forming around the paper disc indicating the ability of the yeast to assimilate, or to grow on, that particular carbohydrate source. Sterile carbohydrate discs are available which can be incorporated into an identification system. With this system, eight or twelve different carbohydrate discs can be delivered in one application to a standard 100.times.15 mm petri plate of inoculated yeast assimilation medium. The disadvantages of this method are the necessity of a boiling bath to melt the agar medium, prolonged technician time, cumbersome storage, and the long time-to-positivity. Additionally, the temperature of the molten agar is typically between 40.degree. and 45.degree. C. at the time of yeast suspension in said agar. Temperatures above these temperatures can kill yeast cultures and thus produce false negatives in the assimilation test results.
Another approach to the auxanographic method is illustrated by the A.P.I. 20C System. In this system, a series of twenty wells containing lyophilized carbohydrates and a glass vial filled with the basic yeast assimilation medium, without a pH sensitive dye, is provided. The glass vial is placed in a boiling water bath to melt the agar medium. After cooling, the yeast is transferred to the medium using a sterile wooden applicator stick and the medium is stirred to make a uniform suspension. This suspension is then transferred by sterile pipet to the nineteen carbohydrate wells and allowed to solidify. The inoculated strip of wells is then placed in a plastic incubation tray provided by A.P.I. and incubated at 30.degree. C. This system requires reactions to be read at 24, 48 and 72 hours with a positive reaction determined by increasing turbidity. Disadvantages of this system are the necessity of a boiling water bath to melt the agar medium, technician time, long time-to-positivity, and relative expense. Additionally, the temperature of the melted agar is typically between 40.degree. and 45.degree. C. at the time of yeast suspension in said agar. Temperatures above these temperatures can kill yeast cultures and thus produce false negatives in the test results.
Identification of Cryptococcus neoformans is generally recognized to be more difficult than the identification of Candida albicans in that Cryptococcus neoformans undergoes no morphological changes which can be observed and remains unicellular throughout its growth cycle. Until recently, one or more of three basic tests, or a combination thereof, were employed to identify the presence of the genus Cryptococcus. One of the methods of identification comprises microscopic inspection of a specimen to identify whether or not a capsule-like formation around the cells of the fungi is present. In order to aid in the inspection of such capsule-like formations, a specimen is surrounded with india ink which enhances the appearance of the capsule by providing a clear and translucent image against the black background making such capsules easier to identify during microscopic examination. A second method employed to identify the genus Cryptococcus comprises plating the specimen on a medium containing urea and a color indicator. Because Cryptococcus produces an enzyme known as urease it has the capability to break down and use the nitrogen contained in the urea, causing the pH to rise, thereby changing the color of the indicator. Therefore, metabolism of a urea containing medium is indicative of the presence of Cryptococcus, Trichosporon beigelii or Candida krusei. A third method of identification of the genus Cryptococcus relies on the ability of that genus to produce a starch-like compound. When the starch-like compound is present, addition of iodine will cause a purple ring to appear around the colony. It is to be noted that none of these tests is specific for Cryptococcus neoformans by itself or in combination, as other species within the genus Cryptococcus and other genera of yeast may also give a positive reaction.
Although the urease test, described above, is not specific for Cryptococcus neoformans, it is a relatively rapid preliminary screen for the genus Cryptococcus which characteristically possess the enzyme urease that is necessary for the hydrolysis of urea. Three methods are currently employed to detect urease production by yeast cultures: (1) urea agar slant, (2) urea broth and (3) urease swab test. The urea agar slants are prepared from commercially dehydrated medium containing a color indicator. After autoclaving, slanting and solidification, the tubes are ready to use. Positive urease reactions are noted by a color change of the medium from orange to pink which occurs within 24 to 72 hours. The primary disadvantages are long time-to-positivity and technician time.
The urea broth test essentially changes the test to a liquid state and increases the inoculum to substrate ratio. The urea broth can be purchased commercially in a lyophilized form and must therefore be reconstituted prior to use. This test requires 3 to 4 hours for positive reactions indicated by a color change of the broth from orange to pink. The major disadvantages are preparation time and desirability of an even more rapid preliminary screen.
The urea swabs are prepared by impregnating sterile cotton applicator swabs with concentrated urea agar base, quick-freezing at -70.degree. C. and lyophilizing overnight. The swabs are inoculated with 2 to 3 yeast colonies, placed into a test medium, the pH of the media is adjusted to pH 4.6, then incubated and examined for a positive reaction indicated by a color change which occurs within 15 to 20 minutes. The basic problem with this test is that the swabs are not commercially available and the pH adjustment for the swab is so critical that there is a likelihood of false negative or false positive reactions occuring.
Perhaps the single most successful and specific conventional test for Cryptococcus neoformans includes the use of bird seed agar. It was discovered that when Cryptococcus neoformans was present in a sample plated on bird seed agar a specific tell-tale brown color would appear within a period of five days to two weeks. This method was improved by using an extract of bird seed which lowered the identification time 3 to 5 days. Later it was discovered that the brown pigment coloration of the yeast was the result of the reaction between the enzyme phenol oxidase and a particular substrate present in bird seed agar. Accordingly, use of substituted phenols such as caffeic acid in the growth medium further shortened the period of time necessary for identification to about forty-eight hours. A still further refinement of the use of caffeic acid to identify Cryptococcus neoformans is set forth in an article by Hopfer and Groschel entitled "Six Hour Pigmentation Tests for the Identification of Cryptococcus neoformans", Journal of Clinical Microbiology, August 1975, Vol. II, No. 2, p. 96-98. The improvement set forth therein includes combining caffeic acid with ferric citrate and incorporating these compounds onto paper discs for use as substrates for the phenol oxidase enzyme activity of Cryptococcus neoformans. Use of these caffeic acid-ferric citrate impregnated paper discs further lowers the identification time to 3 to 6 hours. However, the solution of caffeic acid and ferric citrate used to impregnate the paper discs is quite unstable when exposed to light and temperature and therefore presents serious storage problems. These discs must be stored at -20.degree. C. Furthermore, the relative concentration of caffeic acid and ferric citrate are critical and an unbalanced combination will require longer incubation periods for production of a dark pigment, or, in some cases, nonspecific pigmentation of saprophytic Cryptococcus and several Candida species. In an article entitled "Two Rapid Pigmentation Tests for Identification of Cryptococcus neoformans," Journal of Clinical Microbiology, February, 1982, Vol. 15, No. 2, p. 339-341, by Kaufmann and Merz, the authors present two tests for Cryptococcus neoformans based in part upon modifications of previous approaches to detect phenol oxidase activity. The first test employs corn meal agar containing an emulsifying agent sold under the tradename Tween 80 by Atlas Chemical Company supplemented with caffeic acid The second is a non-medium test utilizing a phenol oxidase detection strip saturated with buffered L-.beta.-3,4-dihydroxyphenylalanine (L-DOPA)-ferric citrate solution. While substrate stability is increased in these tests, storage still presents some problems as ferric citrate, for example, must be stored at -20.degree. C.
Recently, a new culture medium for the identification of Cryptococcus neoformans, Candida albicans and Candida stellatoidea was discovered which includes caffeic acid, oxgall, saponin and a supporting agent. This medium (hereinafter sometimes referred to as original SOC) is disclosed in U.S. Pat. No. 4,144,133, issued Mar. 13, 1979 and entitled "Fungal Growth Media". The phenol oxidase substrate, preferably in the form of caffeic acid, provides for a specific identification of Cryptococcus neoformans by means of the appearance of the characteristic brown pigmentation of the yeast which results from specific enzyme activity of Cryptococcus neoformans on the phenol oxidase substrate. The oxgall, in addition to its known function of suppressing bacterial growth, has also been discovered to enhance filament and chlamydospore production of the medically important fungi Candida albicans and Candida stellatoidea. The purified saponin employed in the fungal media significantly enhances the germ tube and chlamydospore formation of Candida albicans thus providing for rapid identification of these especially serious types of pathogenic fungi. The carrying agent, such as common agar or silica gels for example, simply provides a supporting base for the above described active ingredients.
Although the SOC culture media are specific for identification of Cryptococcus neoformans and rapidly indicate the presence of either the albicans or stellatoidea species of the genus Candida, chlamydospore formation is weak in some strains of Candida albicans requiring 48 to 72 hours for formation rather than 28 to 48 hours for the strong chlamydospore formers.
Thus, while a variety of methods and media have been employed in order to identify and differentiate various fungal pathogens including the critically important genera and species Candida albicans and Cryptococcus neoformans, there is a continuing need for a fungal growth media and system which will rapidly identify and differentiate these genera and species as well as other pathogenic fungi in a rapid, efficacious, economic and technically efficient manner.